Sex Differences in Glomerular Protein Expression in Mice with Autosomal Recessive Alport Syndrome

Background: Alport syndrome (AS) is a monogenic disorder that leads to progressive kidney disease, ear, and ocular abnormalities. It is caused by pathogenic variants in COL4A3, COL4A4, or COL4A5, which encode the α3, α4, and α5 chains of type IV collagen. COL4A3 and COL4A4 reside on chromosome 2q36, while COL4A5 is on the X chromosome. We evaluated for sex differences in mice with autosomal recessive AS. Methods: We compared differences in global protein expression in isolated glomeruli between 1-day old male and female Col4a3 knockout (KO) and wildtype (WT) mice with mass spectrometry (MS). MaxQuant was used for analysis and statistical tests were done on Perseus. Pathway analysis was performed using Gene Ontology. Results: At postnatal day 1 (P1), we observed more severe disease in male compared to female Col4a3 KO mice, as evidenced by higher urine albumin-creatinine ratios (uACR) (1088 mg/mmol vs. 875.6 mg/mmol, p=0.4). 309 significantly differentially expressed proteins in the glomerulus were identified, of which 208 were downregulated and 101 were upregulated in male Col4a3 KO mice. We also compared P1 WT male and female mice and no differentially expressed protein was detected at baseline. Interestingly, the well-known podocyte apical surface transmembrane sialoglycoprotein, podocalyxin, was upregulated in males compared to female Col4a3 KO mice. Pathway analysis showed that Col4a3 KO male mice also had decreased biological processes suggestive of impaired glomerular structure maintenance such as actin filament bundle assembly, cell morphogenesis involved in differentiation and endoplasmic reticulumnucleus signaling pathway. By contrast, pathways including peptide biosynthetic process and ATP biosynthetic process were found to be increased in male Col4a3 KO mice, possibly due to increased compensatory mechanisms. Conclusions: One day old male compared to female Col4a3 KO mice display more severe disease. Podocalyxin has been reported to be excreted in urine due to podocyte loss and we postulate that its upregulation in male Col4a3 KO mice may represent compensation. Overall, glomerular proteomic comparison highlights biological pathways that can explain phenotypic differences between male and female mice with autosomal recessive AS, though its mechanistic drivers are unclear. Funding: Private Foundation Support

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